Hit-to-Lead Optimization of Heterocyclic Carbonyloxycarboximidamides as Selective Antagonists at Human Adenosine A3 Receptor

Antagonism of the human adenosine A3 receptor (hA3R) has potential therapeutic application. Alchemical relative binding free energy calculations of K18 and K32 suggested that the combination of a 3-(2,6-dichlorophenyl)-isoxazolyl group with 2-pyridinyl at the ends of a carbonyloxycarboximidamide group should improve hA3R affinity. Of the 25 new analogues synthesized, 37 and 74 showed improved hA3R affinity compared to K18 (and K32). This was further improved through the addition of a bromine group to the 2-pyridinyl at the 5-position, generating compound 39. Alchemical relative binding free energy calculations, mutagenesis studies and MD simulations supported the compounds’ binding pattern while suggesting that the bromine of 39 inserts deep into the hA3R orthosteric pocket, so highlighting the importance of rigidification of the carbonyloxycarboximidamide moiety. MD simulations highlighted the importance of rigidification of the carbonyloxycarboximidamide, while suggesting that the bromine of 39 inserts deep into the hA3R orthosteric pocket, which was supported through mutagenesis studies 39 also selectively antagonized endogenously expressed hA3R in nonsmall cell lung carcinoma cells, while pharmacokinetic studies indicated low toxicity enabling in vivo evaluation. We therefore suggest that 39 has potential for further development as a high-affinity hA3R antagonist.


■ INTRODUCTION
Adenosine, a naturally occurring purine nucleoside, is an endogenous agonist of adenosine receptors (ARs). 1 ARs are G protein-coupled receptors (GPCRs) comprising four subtypes: A 1 , A 2A , A 2B , and A 3 .While the A 2A R and A 2B R subtypes activate G s to stimulate adenylyl cyclase, increasing 3′,5′-cyclic adenosine monophosphate (cAMP) levels, A 1 R and A 3 R conversely couple to G i/o subunits inhibiting adenylyl cyclase.Beyond inhibiting cAMP accumulation, A 3 R has been suggested to modulate mitogen-activated protein kinase (MAPK) activity which may explain the role of this receptor on cell proliferation and differentiation 2,3 and in tumor development and progression.Evidence suggests that human A 3 R (hA 3 R) antagonists might become new therapeutic tools for the treatment of both chronic renal disease 4 and acute renal ischemia and reperfusion injury. 4Furthermore, hA 3 R antagonists have demonstrated efficacy in eye pathologies. 2Indeed, it has been reported that the potent A 3 R antagonist MRS1220 (N-[9-chloro-2-(2-furanyl)-1,2,4-triazolo [1,5-c]quinazolin-5-yl]benzeneacetamide) prevented oligodendrocyte damage and myelin loss triggered by ischemia or by activation of A 3 R in the rat optic nerve. 5Hence, blockage of hA 3 R has proven to be useful for the treatment of diverse diseases; however, its role is still to be elucidated in other pathophysiological conditions, such as inflammation, cancer, or pain. 2 The identification of new potent and selective ligands which can clarify the therapeutic potential arising from blocking or stimulating the hA 3 R remains an attractive objective. 2,6xperimental structures have been resolved for all the four subtypes of hARs that have become established drug targets, although hA 3 R only has an active structure reported. 7Such experimental structures can help to understand the binding interactions of ARs with ligands and provide templates for structure-based drug design (SBDD) as others 8−14 and we 15 have shown.Free energy perturbation coupled with the molecular dynamics simulation (FEP/MD) method 16,17 has been applied for lead fragment optimization against A 2A R 18, 19 or for structure−activity relationship (SAR) interpretation, e.g., of 3-deazaadenosine agonists 20 and thiazolo [5,4-d]pyrimidines antagonists 21 against A 2A R, or 4-substituted-1,4-dihydrobenzo- [4,5]imidazo [1,2-a]pyrimidine-3-carboxylate antagonist against A 2B R. 22 Furthermore, we have performed the equivalent with FEP/MD thermodynamic integration coupled with the molecular dynamics simulation method (TI/MD) 23 to describe accurately the structure−affinity relationships of antagonist of human A 1 R (hA 1 R).
In a previous work, from virtual screening (VS) of an ∼18,000 compound library, we identified hits of different structures as antagonists of ARs with a new structure having low micromolar affinities using radiolabeled assays. 15Of particular interest for further development were the hits K5, K9, K10, K11, K15, K17, K18, and K32 (Scheme 1) that are heterocyclic carbonyloxycarboximidamide derivatives. 15,24,25These hit compounds (which we purchased from commercial libraries but are synthetically feasible) showed selective low micromolar affinity against hA 3 R using radiolabeled assays, and we showed that the affinities were, in most cases, consistent with antagonistic receptor activities determined using inhibition of cAMP accumulation. 25electing ligands based on their affinity, an equilibrium parameter, does not necessarily predict in vitro activity, and a ligand's kinetic properties may provide a better indication of how a ligand will perform in vivo.Kinetic profiling in the drug discovery process allows the resolution of ligand−receptor interactions into both molecular recognition (dependent on association rate constant K on ) and complex stability (dependent on ligand's dissociation rate constant K off ).Significantly, this enables estimates of the residence time (RT = 1/K off ) of that ligand upon its target. 26By testing the compounds shown in Scheme 1, we observed 15,24,25 that adding chlorine atoms in the phenyl ring of compound K5 increased affinity and antagonistic potency in K17 and K18.An additional interesting finding was that replacement of the five-membered thiazole ring of compound K17 with the six-membered pyridine ring maintained the binding affinity in K10, K11, and K32, as well.
Here, we presented a hit-to-lead study through SBDD using TI/MD 27,28 for the calculation of relative binding free energies and a previous SAR study. 15,24,25The accuracy of perturbative binding free energy methods for ligands AR or other class A GPCR systems was previously shown using the FEP/ MD 20,22,29−31 as well as by TI/MD calculations on complexes of A 1 R. 32 Both of the FEP/MD 33 and TI/MD 27,28 methods can provide accurate results for relative binding free energies with a method error of 1 kcal mol −1 . 28he SBDD and synthesis led to 25 new compounds (37−61) which we tested for their affinity at hA 3 R using nanolucifereasebased bioluminescence resonance energy transfer (NanoBRET) binding assay.Among these, seven compounds 37, 39, 40, 47,  48, 59, and 60 displayed similar or significantly higher affinity than K18.Their binding kinetic parameters including K on , K off , and RT were determined, as well as the hAR subtype selectivity.Compounds 37 and 39 showed ∼10-fold increased potency against hA 3 R and ∼20-fold higher RT compared to K18 or K32.Based on 37 and 39, we explored the tolerance of the chlorine atoms in the 2,6-dichlorophenyl group by synthesizing and testing 4 more new analogues (74−77) with bromine or methyl groups instead and found that 39 still showed the highest affinity and selectivity toward hA 3 R.We then applied the TI/MD method 27 to confirm quantitatively with an accurate method the observed structure−affinity relationships.We performed 500 ns MD simulations of the representative compounds 37−39, 56− 57, and 60 to describe the interactions with residues in the orthosteric binding pocket and conducted an exhaustive analysis with in vitro mutagenesis experimentation for our lead compound 39 in comparison with 37 that also informs for SARs 26,41 Further, since hA 3 R has often been referenced as a promising target for treating cancer, 34 we explored the ability of 37 and 39 to selectively antagonize hA 3 R in a nonsmall cell lung Scheme 1.Chemical Structures, Dissociation Constants with Radio-Labeled Assays (K i in μΜ), and Antagonistic Potencies (pA 2 ) of K18/K32 Analogues Reported in Ref 25; n.a.,Not Active

Journal of Medicinal Chemistry
Figure 1.500 ns MD simulations for the complex of compound K18 with the wild-type (WT) hA 3 R using the amber ff19sb. 39(A) Representative frame of K18 inside the orthosteric binding area; (B) receptor−ligand interaction frequency histograms; bars are plotted only for residues with interaction frequencies ≥0.2.Color figure in frames or bar plots: ligand is shown with pink sticks and ligand's starting position with a pink wire, receptor is shown with a white cartoon and sticks, hydrogen bonding interactions are shown with yellow dashes or bars, π−π interactions are shown with green dashes or bars, hydrophobic interactions are shown with gray bars, and water bridges are shown with blue bars.(C) Root-mean-square deviation (rmsd) plots of Ca carbons of the protein (gray line) and of heavy atoms of the ligand (magenta line).For MD simulations, we used a revised model of the inactive form of hA 3 R we have recently published, 40 generated using the multistate Alphafold 2(AF2) method 41,42 of hA 3 R generated from GPCRdb web-tool; 43 the complexes of the starting structure (docking pose) and final snapshot from the MD simulations are available as pdb files (see https://github.com/annachor/inactive_A3R_AF2-carbonyloxycarboximidamides_MDs).
cancer cell line that endogenously expresses all 4 AR subtypes. 35he ability of 39 to inhibit cancer cell proliferation led us to perform a preliminary pharmacokinetic study, which displayed good lipophilicity and permeability across intestinal cells but a relatively low aqueous solubility and metabolic stability.We therefore present the high-affinity hA 3 R antagonist 39 as a new lead compound for future development.

■ RESULTS
Structure-Based Drug Design.Previously using mutagenesis experiments, and MD simulations using the amber14sb force field (ff14sb) 36 or OPLS2005 37 with Poisson−Boltzmann or generalized Born and surface area continuum solvation (MM/PBSA 38 MM/GBSA calculations 38 ) binding free energy calculations, 24,25 we have suggested a preferred binding pose for K18 or K17 bearing the 1,3-thiazolyl and 2,6-dichlorophenyl or 2-chlorophenyl groups, respectively, and K32 (compound 42 in this study) having the 2-pyridinyl and 2-chlorophenyl groups at the two ends of the carbonyloxycarboximidamide linker.Τhe binding pose (after 500 ns MD simulations with ff19sb, 39 ) for compound K18 is shown in Figure 1A.
In this binding pose, the dichlorophenyl group in K18 is oriented toward transmembrane (TM) 5 and TM6, instead of TM1 and TM2, thus interacting with V169 5.30 and I249 6.54 .This preference was increased with the number of chlorine atoms (as is reflected by the binding affinity constants of the compounds K18, K17, and K5 15,24,25 (Scheme 1).Compared to 1,3thiazolyl in K17, the more basic 2-pyridinyl group 44 in K32 could form a stronger hydrogen bonding interaction with N250 6.55 leading to a ∼2-fold higher affinity of K32. 15,24,25eplacement of isoxazole in K5 with a phenyl group in K39 reduced the binding affinity and replacement of thiazolyl or pyridinyl in K17 or K10, K11, and K32 by phenyl in K15 also reduce their binding affinity. 24,25ased on these observations, we assumed that in compound 37 (Scheme 2), the combination of the 2,6-dichlorophenyl and the 2-pyridinyl groups at the ends of the carbonyloxycarboximidamide linker would enhance affinity.To quantify these predictions using SBDD, we performed TI/MD simulations with ff14sb 39 in heterocyclic carbonyloxycarboximidamide− hA 3 R complexes embedded in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers.We used for the MD simulations a revised model of the inactive hA 3 R generated based on the multistate AF2 method 41,42 that we recently published. 40Interestingly, in the experimental structure of the active hA 3 R (PDB IDs 8X16, 8X17 7 ), the orientation of the M172 5.33 , R173, 5.34 and M174 5.34 motifs, which we showed in ref 40 as important for the residence time of antagonists, matches the conformation we adopted in our revised 40 multi-AF2-based model for the inactive hA 3 R (Figure S1), which we used for all the MD simulations in the present work.We performed the calculations using a 1-step protocol which changes the partial charges and the van der Waals interactions in a single simulation by activating both Lennard-Jones and Coulomb softcore potentials simultaneously.
Indeed, our TI/MD calculations suggested that the alchemical transformation K18 → 37, where 1,3-thiazolyl is changed to 2-pyridinyl, was favored by a relative binding free energy (ΔΔG b,TI/MD ) equal to −0.77 ± 0.08 kcal mol −1 .Also, the alchemical transformation 44 → 37 that changes the 2chlorophenyl to 2,6-dichlorophenyl was favored by ΔΔG b,TI/MD = −1.83± 0.06 kcal mol −1 .A striking observation from our previous experiments 25 demonstrated that mutation of L90 3.32 in the low region of the receptor area, which makes a direct interaction with K18, and the remote L264 7.35 in the middle/ upper region (both to alanine) increased the antagonistic affinity of K18.This suggests an available empty space in these regions of the orthosteric area that can be filled with a sizable hydrophobic group for increasing the ligand's affinity.Based on the commercially available synthetic fragments, we tested the addition of a bromine atom at the 5-position of the 2-pyridinyl group in 37 so that it could fit into the bottom area of the receptor.Thus, using TI/MD calculations, we observed that the alchemical transformation 37 → 39 where a bromine group was added was favored by ΔΔG b,TI/MD = −0.67 ± 0.04 kcal mol −1 .However, the addition of two chlorine atoms at 3,5-positions of the 2-pyridinyl group in 37 was not favored since for the alchemical transformation 37 → 38, we calculated ΔΔG b,TI/MD = 0.58 ± 0.04 kcal mol −1 .
We performed additional TI/MD calculations to determine whether replacement of the 2-pyridinyl group in compound 37 by other groups could further improve affinity.For this purpose, we decided to introduce a methyl group at position 6-of the pyridine ring of 37 to obtain 40, or transfer its nitrogen atom to obtain the 3-pyridinyl analogues 46−48.In addition, we replaced the pyridine ring with the pyrimidin-2-yl moiety to obtain derivative 45, with the 3-tolyl group to obtain compound 49 or with a bulkier, nitrogen-containing, bicyclic system such as the indole or the purine ring, leading to compounds 52 and 53, respectively.Finally, we introduced a methylene linker between the carbonyloxycarboximidamide moiety and the pyridine ring to obtain analogues 54 and 55.In order to establish our previous observations, 15,24,25 with compounds K5, K17, and K18, which suggested that by increasing the number of chlorine atoms on the phenyl ring of the isoxazole group the binding activity was increased, we decided to study selected analogues bearing one chlorine atom or unsubstituted on this phenyl ring (compounds 41−44 and 50−51).Thus, we performed TI/MD binding free energy calculations for most of the above-mentioned alchemical transformations (Table S1).Even when the alchemical calculations did not suggest improved binding to hA 3 R, we still felt that their synthesis was required since it would provide further experimental validation (using in vitro pharmacological techniques) of our model.We therefore synthesized compounds 37−55 (Scheme 2).
Furthermore, in compounds 56−61 (Scheme 2), we sought to investigate the effect on the antagonistic activity caused by the incorporation of the carbonyloxycarboximidamide pharmacophore between the 2,6-dichlorophenyl isoxazole and the aromatic nitrogen heterocyclic ring, into a rigid 1,2,4-oxadiazole ring.This transformation might be achieved through ring closure of the corresponding carbonyloxycarboximidamide analogues.The 500 ns MD simulations with ff19sb 39 suggested that compound 56, the cyclic analogue of compound 37, is stabilized inside the orthosteric binding area through hydrogen bonding interactions between the amide side chain of N250 6.55 and the pyridinyl and 4-oxadiazolyl nitrogen atoms.
All the above-mentioned aryl or aralkyl carbonitriles were treated with hydroxylamine hydrochloride in the presence of a base or aqueous hydroxylamine and were converted to the aryl amidoximes 18−30 (Scheme 4 Table S2).The amidoximes reacted with the acyl chlorides 34−36, easily prepared from the commercial carboxylic acids 31−33, respectively, to result in the corresponding target derivatives 37−55.
In Vitro Pharmacological Characterization.Quantifying the Binding Affinity and Kinetics of Potential Antagonists at hA 3 R.We have previously reported the characterization of hit compound K18, a specific hA 3 R (K i < 1 μM) competitive antagonist with a new scaffold as hA 3 R ligand. 15,25Based on this scaffold, 25 analogues were synthesized to further investigate the SAR, aiming to develop A 3 R antagonists with high affinity as well as high specificity.The compounds were grouped A−D based on their backbone (Tables 1, S3).
As we 25 and others 46 previously showed, NanoBRET ligand binding assay can also be used to determine the parameters of the ligand binding kinetics.We have previously determined the binding kinetic parameters of fluorescent ligand CA200645 with K on (k 1 ) = 3.25 ± 0.03 × 10 6 M −1 min −1 and K off (k 2 ) = 0.019 ± 0.003 min −1 . 25Using the "kinetics of competitive binding" model (built in Graphpad prism 9.3.1),we determined the K on (k 3 ) and K off (k 4 ) of the new seven compounds (Table 2).From these parameters, the RT was determined as 1/K off (Table 2).As shown in Table 2, among these seven compounds, 39 showed largest K on (5.95 ± 0.42 M −1 min −1 ) and the smallest K off (0.046 ± 0.002 min −1 ) and therefore longest residence time (22.1 ± 1.0 min), which contributed to its high binding affinities.
Seven High-Affinity Antagonists Displayed High Selectivity for hA 3 R over the Other AR Subtypes.To assess the competitive antagonistic action of the seven most potent compounds 37, 39, 40, 47, 48, 59, and 60 at all human AR subtypes, cAMP accumulation assays were performed in CHO-K1 cells stably expressing the individual receptors, hA 1 R, hA 2A R, hA 2B R, or hA 3 R. Increasing concentrations of the nonselective AR agonist 5′-N-ethylcarboxamidoadenosine (NECA) and 10 μM antagonist or DMSO control were coincubated for 30 min.Also, for the G i/o -coupled hA 1 R and hA 3 R, 1 μM forskolin (a plant toxin that activates adenylyl cyclase independent of the G protein) was added to stimulate cAMP production.For the G scoupled hA 2A R and hA 2B R, forskolin was not added since the receptors are able to stimulate cAMP accumulation alone.As shown in Figure 3 and Table S4, among these 7 compounds, 37, Scheme 3. Preparation of Carbonitrile 17 a a Reagents and conditions: (a) (i) 1.4 equiv of NaH, DMF dry, 0 °C, 1 h, (ii) 1.5 equiv of iodomethane, room temperature (rt), 20 h, 58% for 15 and (b) 0.11 equiv of Zn, 0.6 equiv of Zn(CN) 2 , 0.02 equiv of Pd 2 (dba) 3 , 0.04 equiv of dppf, DMA, reflux, 3 h, 79%.All the equilibrium binding affinities (pK i ) were determined with NanoBRET ligand binding assay and represented as mean ± standard error of the mean (SEM) of n independent repeats with experiment conducted in duplicates.Data of K18 was taken from ref 25.One-way ANOVA with Dunnett's post-test was used to determine the statistical significance (*p < 0.05) compared to the pK i of K18.39, 40, 47, and 48 selectively antagonize hA 3 R but not the other AR subtypes.Apart from strong potency at hA 3 R, 60 showed weak antagonism at hA 2A R and hA 2B R, while 59 had weak antagonistic effects at hA 1 R, hA 2A R, and hA 2B R.
For the most potent compound 39, different concentrations of the antagonist were used at hA 3 R to perform a full Schild regression analysis (Figure S2).From this analysis, the resulted estimation of antagonist affinity (pA 2 ) was 7.95 ± 0.15, and the Schild slope was found to be close to unity, indicating that 39 acts as a competitive antagonist at hA 3 R.
Further Exploration of the Role of the 2,6-Dichlorophenyl Group in the Binding Affinity toward A 3 R.All seven highaffinity antagonists shared a common structure of the 2,6dichlorophenyl group at position 3 of the isoxazole ring, and the decrease of the number of chloro-substituents leads to reduction in binding affinity (compounds of groups B and C, Table S3).Therefore, we further explored the importance of these two chloro-groups by synthesizing the structural analogues of our most active lead compounds 37 and 39, where the two chlorogroups were replaced by either bromo-or methyl-groups, leading to the corresponding derivatives 74−77 (Scheme 6).The results from TI/MD calculations for the alchemical transformations 37 → 74 or 37 → 76 that change 2,6dichlorophenyl to 2,6-dibromorophenyl or 2,6-dimethylphenyl were ΔΔG b,TI/MD = −0.53± 0.04 or −0.64 ± 0.05 kcal mol −1 , respectively.The corresponding alchemical transformations for 39, 39 → 75, or 39 → 77 were ΔΔG b,TI/MD = −0.45± 0.04 or −0.46 ± 0.04 kcal mol −1 , respectively.The calculations suggested a possible small improvement in binding affinity, although the error of the method is 1 kcal mol −1 which corresponds to an ∼5-fold difference in binding affinity.
Nevertheless, we synthesized compounds 74−77 (Scheme 6) and then characterized these 4 new compounds using NanoBRET ligand binding assay at hA 3 R to determine both their affinities and kinetic parameters (Tables 3 and 4).The synthesis of the novel derivatives 74−77 was performed starting from commercially available 2,6-disubstituted benzaldehydes 62 and 63 that were converted to the isoxazole methyl esters 68 and 69, respectively, upon oxime formation, chlorination with Nchlorosuccinimide (NCS), and subsequent ring closure of intermediates 66 and 67 with methyl acetoacetate. 47The methyl esters 68 and 69 were hydrolyzed under basic conditions to afford the carboxylic acids 70 and 71 that were converted to the corresponding acyl chlorides 72 and 73.Finally, the latter were coupled with amidoximes 18 and 20 to afford the target derivatives 74−77 (Scheme 6).The substitution to bromine from chlorine significantly increased the affinity of 37 but reduced the affinity of 39, whereas the substitution to the methyl group reduced the affinities of both compounds significantly.However, these compounds still displayed equal or higher affinity when compared with K18.Also, we employed cAMP accumulation assay to study their subtype selectivity.These four compounds maintain the hA 3 R specificity observed for their precursors 37 and 39 (Table S5 and Figure S3), all failing to antagonize hA 1 R, hA 2A R, and hA 2B R. Finally, interspecies differences of adenosine A 3 R are higher than the other ARs, and this results in the difficulties in developing A 3 R antagonists which have cross-species activity. 48For completeness, we examined the affinity of the 11 compounds 37, 39, 40, 47, 48, 59, 60, 74, 75, 76, and 77 at rat A 3 R (rA 3 R) using the NanoBRET ligand binding assays using Nluc-rA 3 R. None of the   compounds show affinity K i > 1 μM at Nluc-rA 3 R, suggesting that all are species selective (Figure S4).
As summarized in Table 5, the affinities of these 11 compounds determined from (a) the equilibrium binding affinity measured in NanoBRET assay (pK i ), (b) the kinetic dissociation constant (pK d ), and (c) the functional dissociation constant (pK B /pA 2 ) using either the dose ratio eq 1 or for 39 the Schild regression analysis showed excellent agreement, confirm-  All the equilibrium binding affinities (pK i ) were determined using NanoBRET binding assay and represented as mean ± SEM of n independent repeats with experiment conducted in duplicates.Data of K18 was taken from ref 25.One-way ANOVA with Dunnett's post-test was used to determine the statistical significance (*p < 0.05) compared to the pK i of K18 (p1) and 37 # or 39 ∧ (p2) as appropriate.
ing that these 11 compounds are high-affinity hA 3 R-selective antagonist candidates, especially the lead compounds 39.
Binding Profile Investigation.MD Simulations.To investigate the binding profile of the best antagonists shown in Scheme 2 at hA 3 R, we performed MD simulations with representative compounds, i.e., 37, 38, 39, and 56 in complex with hA 3 R embedded in POPC bilayers.We used a multistate AF2 method 41,42 of hA 3 R generated with the GPCRdb 43 webtool.We optimized this model of hA 3 R in ref 49 to achieve best agreement with thermodynamic and kinetic data.In the starting docking pose of these compounds, the dichlorophenyl group was oriented toward TM5 and TM6 and is the same as we previously observed for K18 and K32 (compound 44 in this study). 15,24,25sing 500 ns MD simulations of the antagonists 37 and 39 in complex with hA 3 R, we were able to reveal the important residues in the orthosteric binding area essential for 37 and 39 binding (Figure 4).We observed that with compound 37, its dichlorophenyl group orientates toward TM5 and TM6 to form dispersion interactions interacting with V169 5.30 , M172 5.33 , and I249 6.54 , F182 5.43 , and I253 6.58 .Moreover, the isoxazole forms aromatic π−π stacking interaction with the phenyl group of F168 5.29 (Figure 4A,B).The amide side chain of N250 6.55 is suggested to form a bidentate hydrogen bond with the carboximidamide amino group, while the 2-pyridinyl nitrogen can form water-bridged interactions with N250 6.55 .Nitrogen and oxygen atoms of isoxazole can form hydrogen bonds with the NH groups of F168 5.29 or V169 5.30 , and the carboximidamide carbonyl group is suggested to form water-bridged interactions with H272 7.43 .There are also other water-bridged interactions between the isoxazol-4-carbonyloxycarboximidamide and example V72 2.64 , F168 5.29 , M172 5.33 , L246 6.51 , and I268 7.39 .The 2pyridinyl group forms dispersion interactions with L90 3.32 , L91 3.33 , T94 3.36 , M177 5.38 , F182 5.43 , W243 6.48 , L246 6.51 , and I253 6.58 .The methyl group in the isoxazole ring can interact with V72 2.64 , Y15 1.35 , A69 2.61 , V72 2.64 , L264 7.35 , I268 7.39 , and H272 7.43 through dispersion interactions.By modifying 37 into 39 through a 5-bromo substitution to the 2-pyridinyl group, we observed that 39 is tilted from TM7 and ΤΜ4 to TM5 showing additional dispersion interactions with M177 5.38 , V178 5.39 and loosing interactions with F168 4.52 , L264, 7.35 and H272 7.43 (Figure 4D,E).Significantly, compared to 37, 39 moves deeper into the bottom of the binding area forming hydrogen bonding interactions with T87 3.29 .In 38, the 3,5-dichloro substitution in the 2-pyridinyl group diminishes affinity by ∼316-fold, and the 500 ns MD simulations suggested that this ligand escaped from the binding area (Figure S5A−C).We further expanded the MD simulation analysis in the oxadiazole series 56−61 by studying compounds 56, 57, and 60 (see Discussion in the Supporting Information and Figure S6).
TI/MD Calculations.MD simulations can describe qualitatively SARs based on the inspection of MD simulation trajectories and protein−ligand interaction frequency plots.While, the TI/MD simulations can accurately calculate the changes in binding affinity between different substituents.The set of the studied compounds K18, 37−42, 44−46, 48, 52, 54, 55, and 74−77 display 3 orders of magnitude differences between their affinity range.The results from the TI/MD alchemical calculations for 23 pairs of ligands in complex with hA 3 R using our optimized model of hA 3 R are shown in Table S1.As is shown in Figure 5, the correlation coefficient between the TI/MD calculated relative binding free energies and experimental values was r = 0.68 (p = 0.026) with mean unsigned error (MUE) = 0.89 kcal mol −1 (Table S1).
Mutagenesis Studies of Compound 39 at hA 3 R in Comparison with 37. Based on the interaction frequency of the amino acid residues in the orthosteric binding area in contact with antagonist, (Figure 4B,D) predicted from the 500 ns MD simulations, we next employed mutagenesis (alanine substitution except where alanine was present, then glycine was used) with NanoBRET binding assay to experimentally investigate residues that were suggested to be important for the binding of 37 and 39.First, since mutation of residues in GPCRs can have a detrimental effect on receptor trafficking, we initially determined the cell−surface expression of WT and mutant Nluc-hA 3 R using fluorescence-activated cell sorting (FACS) in flow cytometry 50,51 and presented as % WT (Table S6). 4 out of 24 of the hA 3 R mutants showed significantly reduced cell surface expression (compared with the WT).Conversely, the hA 3 R mutants V72 2.64 A, F168 5.29 A, and F182 5.43 A displayed increased cell surface expression.Despite these changes in the cell surface expression levels, all the mutants expressed sufficiently to enable NanoBRET ligand binding experiments to be formed.The equilibrium dissociation constant (K d ) of the fluorescent ligand CA200645 was determined for each mutant and the WT hA 3 R�all expressed individually in HEK293T cells (Table S6).While the affinity of CA200645 remained unchanged for most of the hA 3 R mutants, Y15 1.35 A, L91 3.33 A, M172 5.33 A, M177 5.38 A, L246 6.51 A, and I268 7.39 A did display significantly lower affinity for CA200645 with the worst L246 6.51 A (K i ∼ 258 nM) being about 10-fold lower when compared with the WT (∼24 nM). a K on (k 3 ) and K off (k 4 ) for each compound determined using NanoBRET binding assay at Nluc-hA 3 R and fitted with the "kinetics of competitive binding model".RT as determined by 1/K off .Saturation binding affinity (pK i ) determined through NanoBRET competition binding assay using the Cheng-Prusoff equation.Data taken from Tables 1 and 3. b Kinetic dissociation constant (pK d ) for each compound determined from K off /K on in Tables 2 and 4. c Dissociation constant (pK B ) as determined through cAMP response using dose ratio equation for each compound or # Schild analysis for 39.

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For hA 3 R, F168 5.29 A, N250 6.55 A, and H272 7.43 A, CA200645 was unable to bind, so these three could not be further investigated in competition binding assays.Importantly, the reductions in the binding affinity of CA200645 did not correlate with the changes in the cell surface expression, indicating the sufficient expression of all mutants.
In the NanoBRET competition binding assays, 37, 39, and agonist NECA were tested at WT and the 21 mutant hA 3 R, and For MD simulations, we used a revised model of the inactive form of hA 3 R we have recently published, 40 generated using the multistate AF2 method 41,42 of hA 3 R generated from GPCRdb web-tool; 43 the complexes of the starting structure (docking pose) and final snapshot from the MD simulations are available as pdb files (see the Ancillary Information).
the mutational effect was represented as the change in affinity compared with the WT (ΔpK i ) (Table S6 and Figure 6).There were 8 mutants (Y15 1.35 A, L91 3.33 A, M172 5.33 A, M177 5.38 A, V178 5.39 A, F182 5.43 A, L246 6.51 A, and I268 7.39 A) which showed significantly reduced affinity for both 37 and 39, while V65 2.57 A and L90 3.32 A displayed increases in affinity for both antagonists.This indicated the importance of these residues in composing the orthosteric binding pocket of 37 and 39.Noteworthy, while the effects of mutating the residues Y15 1.35 , L91 3.33 , M172 5.33 , M177 5.38 , L246 6.51 , and I268 7.39 to alanine was the same between 37, 39, and CA200645, the residues V178 5.39 and F182 5.43 appear to be unique for 37 and 39 forming dispersion interactions with the 2-pyridinyl group of these antagonists.
Also, 37 and 39 displayed different extents of changes in the affinities at several mutants which might explain the increased affinity of 39 when compared to 37. Compound 39 showed greater reductions in the affinity at L91 3.33 A and L246 6.51 A (∼97-fold and ∼115-fold, respectively) when compared with 37 (∼58-fold and ∼22-fold, respectively).This correlates well with the simulation results (the additional Br going deeper into the binding pocket composite of L91 3.33 A and L246 6.51 A and therefore increasing the affinity).Additionally, W243 6.48 A showed significant reduction (p = 0.0006) in the affinity of 39 but not of 37 (p = 0.2192) when compared with the WT, indicating that there is a stronger π−π interaction of 39 with the extra bromine.On the other hand, I253 6.58 A at the top of the binding pocket had significant reduced affinity for 37 (p = 0.0030) but not for 39 (p = 0.7289), suggesting that 37 interacts more with the top of the binding pocket than 39.
For the completeness of the study, we have also assessed the binding affinity of agonist NECA at all the hA 3 R mutants and compared with WT.Among the 21 mutants tested, there were 12 mutants previously studied with NECA using cAMP accumulation assay. 52The mutational effects of these mutants in the potency of cAMP responses induced by NECA showed a good agreement with their effects at the change of binding affinity of NECA in this study.For example, T94 3.36 A, L246 6.51 A, I268 7.39 A, and M177 5.38 A showed significant reduction in the binding affinity of NECA, while M177 5. 38 A also showed reduction in the cAMP response potency, and the other three mutants showed no cAMP response.Also, the agonist NECA and the antagonists 37 and 39 showed differences in the change of affinity at several mutants.37 and 39 showed increased affinity at both V65 2.57 A and L90 3.32 A while NECA displayed no changes.Also, NECA had higher affinity at I249 6.54 A and lower affinity at V72 2.64 A and T94 3.36 A, while 37 and 39 had no change at these mutants.The agreement between two studies with different types of pharmacological assays and the ability to reveal the differences between the binding pockets of agonist and antagonisshowed the robustness of the mutagenesis study.

Disease Model Validation and In Vitro Pharmacokinetic Profiling. Validation of Compound Selectivity at Endogenously Expressed Receptors in a Disease Model of
Lung Cancer.We next sought to confirm that the lead compounds, 37 and 39, were able to selectively antagonize hA 3 R when endogenously expressed.We utilized LK-2 and NCI-H1792 cells, which are both nonsmall cell lung carcinoma cells.LK-2 cells display low hA 1 R and hA 2B R expression, whereas NCI-H1792 cells express all the 4 adenosine receptor subtypes. 35Neither N 6 -cyclopentyl-adenosine (CPA; was able to inhibit forskolinmediated cAMP accumulation in LK-2 cells, consistent with their low adenosine receptor expression (Figure S7).CPA inhibited forskolin-mediated cAMP production in NCI-H1792 cells (pEC 50 6.51 ± 0.63), but neither 37 nor 39 was able to antagonize the response (pEC 50 values of 6.83 ± 0.52 and 7.13 ± 0.60, respectively) (Figure 7 and Table S7).The response to IB-MECA was more potent (pEC 50 of 8.50 ± 0.32), with both 37 and 39 significantly reducing the potency of the response (6.69 ± 0.57 and 6.24 ± 0.42, respectively).Calculated pK B values were similar to those measured in the heterologous expression system (7.17 ± 0.13 and 7.46 ± 0.20).This confirmed the ability of both compounds to selectively antagonize hA 3 R when endogenously expressed alongside other adenosine receptors.
hA 3 R has often been referenced as a promising target for treating cancer. 34We therefore aimed to see the effect of  S1) for hA3R using NanoBRET binding assay; r: correlation coefficient, s: slope.For TI/MD simulations, we used a revised model we recently published 40 of the inactive form of hA 3 R generated based on the multistate AF2 method 41,42 of hA 3 R. selectivity antagonizing the receptor on cell proliferation.Compound 37 significantly impaired proliferation, displaying toxicity even in the absence of A 3 R (Figure S7 and Table S8).While 39 still reduced proliferation in LK-2 cells, showing some nonspecific toxicity, the effect was increased in NCI-H1792 cells, with an increase in potency and an increase in the percentage of cell death (Figure 7).
Pharmacokinetic Assessment of Lead Compound 39.The highest affinity compound 39 was evaluated in the in vitro pharmacokinetic study including solubility, absorption, and metabolism (Table 6).For the solution properties, 39 showed low aqueous solubility with a mean solubility of 0.10 μM in phosphate buffer solution (PBS), 0.16 μM in simulated gastric fluid, and 43.13 μM in simulated intestinal fluid.It displayed a comparable partition coefficient (Log D = 2.82) with the reference compounds haloperidol (Log D = 2.49) and phenytoin (Log D = 2.28), showing adequate lipophilicity.Compound 39 also showed high protein binding in human plasma, with 99% of the proteins bound, which is similar to that of the reference compounds sertraline (99%) and warfarin (95%).
The in vitro absorption of 39 was assessed using the bidirectional permeability assay (P app ) in Caco-2 cells.Compound 39 had a P app of 7.2 × 10 −6 cm/s from the apical side (A) to the basolateral side (B) and a P app of 0.6 × 10 −6 cm/s from B to A, resulted in an uptake ratio (P app A-B/P app B-A) of 12, Figure 6.Changes in the binding affinities for compounds 37 and 39 and NECA measured using NanoBRET binding against WT and mutants hA 3 R.The binding affinity of 37 and 39 and NECA at hA 3 R WT and mutants were determined using NanoBRET binding assay performed in HEK293T transiently transfected with each construct.The change in affinity (ΔpK i ) is calculated as the difference of pK i between the mutant and WT.Data is represented as mean ± SEM of n = 3 independent repeats conducted in duplicates.Statistical significance (*p < 0.05) compared with WT was determined using one-way ANOVA with Dunnett's post-test.
suggesting no drug efflux across the membrane occurred.However, the percent recovery was lower (26% A to B and 19% B to A) compared to the reference compound in both directions (74% A-B and 96% B-A for propranolol), indicating potential problems like poor solubility or nonspecific binding during the assay.
The in vitro metabolism of 39 was assessed as its intrinsic clearance in human liver microsomes.Compound 39 had a shorter half-life (9.5 min) than the precursor compound K18 (24 min 25 ) as well as the reference compounds (terfenadine, 16.9 min and verapamil 26.6 min).The resulted intrinsic clearance (CL int ) was 729 μL/min/mg of microsome, which was higher than K18 (287.2 μL/min/mg of microsome 25 ) terfenadine (410.1 μL/min/mg of microsome) and verapamil (261.1 μL/min/mg of microsome).These indicated that 39 has a relatively rapid metabolism in hepatic microsomes and therefore will cause less accumulation and potential toxicity.

■ DISCUSSION
Here, we have reported a hit-to-lead study by computational design, synthesis, and screening using NanoBRET binding assay of 25 novel derivatives targeting selectively hA 3 R, as analogues or derivatives of the selective A 3 R carbonyloxycarboximidamide heterocyclic hits K17, K18, 15 or K32 24,25 from a previous structure-based VS. 15,24,25 In these previously reported hits, the carbonyloxycarboximidamide moiety was connected with a 3-(2-chlorophenyl)-isoxazolyl, 3-(2,6-dichlorophenyl)-isoxazolyl or 3-(2-chlorophenyl)-isoxazolyl group, respectively, and the carboximidamide carbon was connected with the 1,3-thiazolyl or 2-pyridinyl group. 15,24,25Their NanoBRET-based determined dissociation constants for K17, K32, and K18 were K i = 600, 250, and 120 nM, respectively, showing the importance of attaching two ortho-chlorine substituents in the phenyl group attached to isoxazole ring and that as regards affinity and antagonistic potency 2-pyridinyl (K32) > 4-(1,3-thiazolyl) (K17) > 3-pyridinyl (K10) > 4-pyridinyl (K11). 24,25However, the residence time of these hits was low and unmeasurable (<1 min). 25e used, for the SBDD of the novel carbonyloxycarboximidamide derivatives, the same binding pose of K18 (or K32) in the orthosteric binding area of hA 3 R.This binding pose was previously suggested 16 after refinement of the docking pose of K18 from VS 15 using 100 ns MD simulations with the OPLS2005 37 force field.The binding pose in K18 14 was further confirmed with a combination of 500 ns MD simulations using the ff14sb and alanine mutagenesis supported by affinity experiments. 24,25In K18 (or K32), the phenyl group that is attached at the 3-position of the isoxazole ring is oriented to the upper region of the receptor, while the 1,3-thiazolyl or 2pyridinyl group was oriented deep in the receptor.There is an increasing propensity of the phenyl group to turn toward TM5 and TM6 by increasing the chlorine substituents attached to the available ortho positions of the phenyl group junction.Based on these observations, we used alchemical binding free energy calculations applied with the TI/MD and ff19sb simulations in complexes of ligands with a revised model for hA 3 R we recently published 40 generated from the multi-AF2-based model for inactive hA 3 R (Table S1) to design and synthesize more potent compounds than K18 (or K32).At the time we submitted this paper, the structure of the active state of hA 3 R in complex with agonists and Gi heterotrimer was reported. 7Interestingly, in the experimental structure, the orientation of R173 5.34 matches the conformation we used in our revised multi-AF2-based model for the inactive hA 3 R that we use for all the MD simulations performed here. 40The purchase of compounds like K18 (or K32) for testing 15,24,25 from companies was costly, and the synthesis of these series with sufficient diversity in structure for exploring SARs was accomplished simply and in good yields using commercially available carbonitriles (Schemes 3 and 4).The heterocyclic carbonyloxycarboximidamides 37−55 and 74−77, and their structurally related 1,2,4-oxadiazole derivatives 56−61, provide novel and highly selective hA 3 R antagonists, which are synthetically feasible and easily accessed drug molecules amenable for further development.
Among the 29 synthesized compounds (belonging in groups A−F; Tables 1, 3, and S3), there are three groups of compounds (A−C; Tables 1 and S3) which only differ by the number of chloro-substituents in the phenyl ring of 3-phenyl-isoxazole, (a) 37, 42, and 44; (b) 40, 41, and 43; and (c) 49, 51, and 50.By comparing their binding affinities at hA 3 R, we showed that the affinities of antagonists increased as the number of chlorosubstituents increased, which is in line with our findings. 15,24,25n agreement with our previous studies, 25 the addition of chlorine atoms at the ortho positions of the phenyl group in the 3-phenyl-isoxazole moiety enhanced affinity since the dichlorophenyl group enhances the hydrophobic interactions toward TM5 and TM6 with residues V169 5.30 and I249 6.54 .This was shown in the relative binding free energy values which are Aqueous solubility (μM) in PBS at pH 7.4/simulated gastric fluid/simulated intestinal fluid determined with high-performance liquid chromatography−ultraviolet spectroscopy.b The partition efficient of the compound between n-octanol and PBS at pH7.4, measuring the lipophilicity of the tested compound.Log D was calculated as Log10(the amount of compound in n-octanol/the amount of compound in PBS).c Measure of percentage of protein binding and percentage of compound recovery during the assay determined in equilibrium dialysis using human plasma.d The permeability of compounds assessed in bidirectional Caco-2 cell permeability assay with pH = 6.5 for donor chamber and pH = 7.4 for receiver chamber.The extent of permeability is measured as apparent permeability coefficient (P app ) from apical (A) to basolateral (B) or in reverse direction.The percentage recovery of the compound is calculated as the total amount of compound in the donor and the receiver at the end of experiment/the amount of initial compound present.The uptake ratio of the compound is calculated as P app A-B/P app B-A. e The metabolic stability of compounds was determined in 0.1 mg/mL human liver microsomes, measured as the half-life (t 1/2 ) and apparent intrinsic clearance (CL int ).
Within group A, all compounds having a dichlorophenyl group attached at the 3-isoxazole position only differ by the aryl group attached to the carbonyloxycarboximidamide moiety, when compared to K18.This aryl group showed crucial effects on the binding affinity.Replacement of 2-pyridinyl group in 37 (Ki = 34.7 nM) with the less basic by ∼10 3 -fold 1,3-thiazolyl group in K18 (Ki ∼ 120 nM) or the less basic by ∼10 4 -fold 2pyrimidinyl group in 45 (K i ∼ 537 nM) led to a reduced affinity by 3.5-fold or ∼15.5-fold due to the stronger hydrogen bond that can be formed between the N250 6.55 amido side chain and 2-pyridinyl group. 44The corresponding relative binding free energy values for K18 → 37 are ΔΔG b,exp = −0.77± 0.08 kcal mol −1 and ΔΔG b,TI/MD = −1.51± 0.08 kcal mol −1 with deviation |ΔΔG b,TI/MD -ΔΔG b,exp | = 0.74 kcal mol −1 and for 37 → 45 are ΔΔG b,exp = 1.69 ± 0.05 kcal mol −1 and ΔΔG b,TI/MD = 0.50 ± 0.04 kcal mol −1 with a deviation of 1.19 kcal mol −1 .
It is worth noting that the five compounds with high affinities in this group all have a nitrogen atom at a similar position as the nitrogen in the thiazole ring of K18, see compound 37, which is in line with our findings. 15,24,25Changes in this nitrogen position reduce the ability of aryl nitrogen to form a hydrogen bond with N250 6.55 amido side chain.Thus, compared to the 2-pyridinyl analogue 37 (Ki = 34.7 nM), the 3-pyridinyl analogue 48 (K i ∼ 100 nM) had ∼3-fold lower affinity similar to previous observations for the monochloro derivatives K32 and K10 (Scheme 1).The corresponding relative binding free energy values for 37 → 48 were ΔΔG b,exp = 0.67 ± 0.05 kcal mol −1 and ΔΔG b,TI/MD = 2.14 ± 0.08 kcal mol −1 with a deviation 1.47 kcal mol −1 .Other changes that reduce the ability of pyridinyl nitrogen to form a hydrogen bond with N250 6.55 amido side chain are discussed below.Replacement of 2-pyridinyl in 37 with 2-pyridinylmethylene in 54 (K i ∼ 316 nM) drives nitrogen away from N250 6.55 and causes reduction in binding affinity, correspondingly, by ∼9-fold.The corresponding relative binding free energy values for 37 → 54 were ΔΔG b,exp = 1.36 ± 0.04 kcal mol −1 and ΔΔG b,TI/MD = 2.55 ± 0.06 kcal mol −1 with a deviation of 1.19 kcal mol −1 .For the same reason is observed reduction in binding affinity by ∼8.7-fold when the 3-pyridinyl group in 48 (K i ∼ 102 nM) is changed to the 3-pyridinyl methylene group in 55 (K i ∼ 891 nM).Compared to 48 (K i ∼ 102 nM) bearing the 3-pyridinyl group, compound 46 (K i ∼ 2089 nM) with the 2-chloro-3-pyridinyl group has 20.5-fold lower binding affinity since the 2-chloro-substituent hampers the hydrogen bonding interaction of 3-pyridinyl nitrogen.This was shown in alchemical perturbation 48 → 46 ΔΔG b,exp = −1.33 ± 0.05 kcal mol −1 and ΔΔG b,TI/MD = −2.64 ± 0.11 kcal mol −1 .The 3-tolyl group in 48 (Ki ∼ 209 nM) does not form a hydrogen bond, and compared to 3-methyl-2-pyridinyl in 40 (Ki ∼ 100 nM), its binding affinity was reduced by 2.1-fold; the corresponding alchemical transformations 55 → 48 showed ΔΔG b,exp = −1.33 ± 0.05 kcal mol −1 and ΔΔG b,TI/MD = −2.64 ± 0.11 kcal mol −1 with a deviation of 1.31 kcal mol −1 and 40 → 49 showed ΔΔG b,exp = 0.30 ± 0.08 kcal mol −1 and ΔΔG b,TI/MD = 1.60 ± 0.09 kcal mol −1 with a deviation of 1.30 kcal mol −1 .Interestingly, while the addition of methyl group at α-position to nitrogen increases pyridine basicity by 5.4-fold, 53 the 6-methyl-2-pyridinyl derivative 40 (K i ∼ 128 nM) has a 3.7-fold lower affinity compared to 37. Therefore, we assumed that the methyl group points toward an area of hA 3 R where the water density is increased.Previously, we suggested 60 that such an area may reside between a polar substituent of the ligand and TM2 and TM3.The corresponding relative binding free energy values for 37 → 40 were ΔΔG b,exp = 0.81 ± 0.06 kcal mol −1 and ΔΔG b,TI/MD = 0.57 ± 0.04 kcal mol −1 , with a deviation of 0.24 kcal mol −1 .
Moreover, a rigidification of the carbonyloxycarboximidamide moiety led to the oxadiazole derivatives with 59 and 60 having slightly higher affinities compared to their precursors 48 and 47.The 500 ns MD simulations showed that rigidification of the carbonyloxycarboximidamide moiety to oxadiazole derivatives 56−61 (class D, Tables 1 and S3) causes reorientation of the dichlorophenyl group through rotation around the oxazoleoxadiazole C−C bond as we showed for compounds 56, 57, and 60 (see Figure S6).Thus, the dichlorophenyl group faces TM2 in compounds 56 and 57 and TM7 in compound 60.Between compounds 56 and 61 (class D, Tables 1 and S3), the 3pyridinyl derivative 59 is a stronger binder by 2-fold compared

Journal of Medicinal Chemistry
to 2-pyridinyl derivative 56, while for the acyloxyimidamide 2pyridinyl derivative, 37 has 2-fold higher affinity compared to 48.Compared to 59 (K i = 91.2nM and RT = 20.5 min), the methyl substituent at 4-position of the 3-pyridinyl group increased the affinity in 60 (K i = 58.9nM and RT = 10.7 min) since is oriented toward M243 6.48 favoring hydrophobic interactions (see Figure S6,G−I), providing an additional lead for further improvement; the TI/MD results for this series are discussed in the Supporting Information.However, in functional cAMP experiments, we observed that they lost their hAR subtype selectivity.This contrasted with compounds 37, 39, 40, 47, 48, and 74−77 which were shown to display high hA 3 R selectivity.Moreover, none of the compounds tested showed high (K i > 1 μM) affinity toward rA 3 R.
We previously determined the effects of receptor mutation on antagonist potency (pA 2 ) for A 3 R L90A 3.32 , V169 5.30 A/E, M177 5.40 A, I249 6.54 A, and L264 7.34 A by functional assays. 25e reported 24,25 that M177 5.38 A caused the most significant reduction on the K18 antagonist effect.Interestingly, we found that L90A 3.32 in the low region and L264A 7.34 in the middle/ upper region increased K18 potency, while I249 6.54 A had little effect (compared to WT hA 3 R). 25We suggested that V169 5.30 was not a selectivity filter for hA 3 R agonists or antagonists.Here, we also showed that 32,44,45 V169 5.30 A did not cause a significant change in affinity for both 37 and 39 (Table S6).Moreover, we did observe significant reductions in affinity for both 37 and 39 with 8 of our mutants hA 3 R (Y15 1.35 A, L91 3.33 A, M172 5.33 A, M177 5.38 A, V178 5.39 A, F182 5.43 A, L246 6.51 A, and I268 7.39 A), while 2 other mutations (V65 2.57 A and L90 3.32 A) produced an increase in affinity for both antagonists 37 and 39, suggesting that these 10 residues comprise the orthosteric binding pocket (Table S6).
However, there are also differences in binding profiles between compounds 37 and 39.Indeed, 39 showed a greater reduction in the affinity at L91 3.33 A (∼97-fold) and L246 6.51 A (∼115-fold) when compared with 37 (∼58-fold and ∼22-fold, respectively).The effects of the Y15 1.35 A, Y265 7.36 , or W243 6.48 A mutations were also more significant for 39 than for 37, which showed excellent agreement with our simulation results.It appears that the sizable hydrophobic bromine can orientate in the orthosteric binding area through the increase in hydrophobic interaction with W243 6.48 and L91 3.33 .This was also validated through analysis of I253 6.58 A which is located on top of the binding pocket.The binding affinity of 37 showed a greater reduction in affinity compared to 39, suggesting that 37 occupies a position near the top of the orthosteric pocket.
As is shown in Figure 5 using an AF2-generated model that we recently published, 40 the correlation between the 23 TI/MD calculated relative binding free energies and experimental values measured with the NanoBRET binding assay was very good with r = 0.68 (p = 0.026) and MUE = 0.89 kcal mol −1 (Table S1).This shows that we can use this procedure for further optimization of this series of compounds which is ongoing research.
Finally, for future optimization and in vivo studies, lead compound 39 was assessed for its affinity for endogenously expressed receptors and in vitro pharmacokinetic properties.Compound 39 selectively antagonized IB-MECA, but not CPA, when looking at the inhibition of cAMP production, demonstrating its hA 3 R selectivity in cells expressing both inhibitory hARs.Furthermore, despite cell toxicity in both cell lines, 39 exerted a greater potency and efficacy for the inhibition of cellular proliferation in lung cancer cells expressing the A 3 R, reinforcing the receptor as a potential target for the treatment of cancer.When looking at the pharmacokinetic suitability of 39, it showed 400-fold higher solubility in simulated intestinal fluid than in PBS or simulated gastric fluid.The partition coefficient and percentage of protein binding and permeability in Caco-2 cells were all comparable to reference compounds haloperidol, sertraline, or labetalol.In terms of in vitro metabolism, compound 39 showed a much larger intrinsic clearance in liver microsomes which resulted in a short half-life (∼9.5 min).Future optimization based on this compound should focus on improving its aqueous solubility and metabolic stability to increase the in vivo efficacy.
■ EXPERIMENTAL SECTION Biological Methods.Compounds.NECA, CPA, IB-MECA, and MRS1220 were purchased from Tocris Bioscience (Wiltshire, UK).Rolipram was obtained from Cayman Chemicals (Michigan, USA).All the ligands above-mentioned were dissolved in DMSO as 10 mM stock and stored at −20 °C until use.Forskolin was purchased from Tocris Bioscience (Wiltshire, UK), made up as 10 mM stock in DMSO, and stored at room temperature.CA200645 and CA200623 were purchased from HelloBio (Bristol, UK), dissolved in DMSO as 100 μM stock, and stored at −20 °C.The purity of the tested compounds was >95% (see Chemistry methods, General Information).
Constructs.The FLAG tag (DYKDDDDK) and Nluc-hA 3 R (gifted by Stephen Briddon) were cloned into vector pcDNA3.1(−).Sitedirected mutagenesis was performed to make A 3 R mutants using the QuikChange Lightning kit (Agilent Technologies, US) according to the manufacturer's protocol.All the construct sequences were confirmed by the DNA sequencing performed by the DNA sequencing facility at the Department of Biochemistry, University of Cambridge (Cambridge, UK).
Cell Culture and Transfection.The source of cells was as described previously. 25,35These cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/Hams F-12 nutrient mix (F12) GlutaMAX media (ThermoFisher, UK), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, Poole, Dorset, UK) and 1% antibiotic-antimycotic (AA) (Sigma, UK).In the cAMP accumulation assay, the Chinese hamster ovary (CHO) K1 cell line was used because of its lack of endogenous ARs expression. 61Stable cell lines of CHO-K1-A 1 R, CHO-K1-A 2A R, CHO-K1-A 2B R, and CHO-K1-A 3 R were cultured with the Hams F-12 nutrient mix (ThermoFisher, UK) supplemented with 10% FBS and 1% AA. 600 μg/mL Geneticin (ThermoFisher, UK) was added to the stable HEK293T and CHO-K1 cell lines for selection, and the medium was changed every 2 days.In the mutagenesis study, HEK293T cells were plated to a 24-well plate and grown overnight.The seeded cells were then transfected with 250 ng of FLAG-Nluc-A 3 R WT or mutant receptor using polyethylenimine 25 kDa (PEI, Polysciences Inc., Germany) in 6:1 ratio, diluted in 150 mM NaCl.LK-2 and NCI-H1792 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% AA.All cells were maintained at 37 °C with 5% CO 2 in a humidified atmosphere.
NanoBRET Binding Assay.NanoBRET ligand competition binding assays were performed to identify the saturation binding affinity (pK i ) and the binding kinetic parameters of the potential antagonists.HEK293 cells stably expressing Nluc-hA 3 R/rA 3 R or HEK293T cells transfected with FLAG-Nluc-A 3 R WT or mutants for 24 h were seeded onto white 96-well plates (Greiner, UK) at a density of 10,000 cells/ well and grown overnight.On the assay day, the medium was discarded and replaced with 100 μL of phosphate-buffered saline (PBS) buffer (ThermoFisher, UK) containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich, Poole, Dorset, UK) and 0.1 μM Nano-Glo Luciferase substrate (Promega, UK).After 5 min of incubation in the dark, tested compounds in different concentrations together with 5 nM CA200645 for Nluc-hA 3 R or 100 nM CA200623 for Nluc-rA 3 R or CA200645 at its Ki concentration for individual FLAG-Nluc-hA 3 R mutants were added.The BRET signal was measured with Mithras LB940, recording the light emission at 460 nM (Nluc) and 610 nM (fluorescent ligands) for 30 min.The raw BRET ratio was calculated by dividing the emission at 610 nM with the 450 nM emission.Nonspecific binding was determined with the addition of high concentration of unlabeled antagonist, 10 nM MRS1220, and the corresponding BRET ratio was used for baseline correction.The baseline-corrected BRET ratio at 10 min poststimulation was used for calculating the affinity constant.
cAMP Competition Assay.In the cAMP inhibition (A 1 R or A 3 R) or accumulation (A 2A R and A 2B R) experiments, CHO-K1 cells stably expressing the corresponding receptor were performed as described previously. 25Briefly, cells expressing the receptor of choice were harvested and resuspended in stimulation buffer (PBS containing 0.1% BSA and 25 μM rolipram) and seeded at a density of 2000 cells/well in the white 384-well Optiplate.Potential antagonists or DMSO control were added with different concentrations of AR agonist NECA.In the experiment with A 1 R/A 3 R, cells were costimulated with 1 μM forskolin (an adenylyl cyclase activator) to detect the inhibition of cAMP response.LK-2 and NCI-H1792 cells were stimulated as above, but using 1000 cells/well, and 0.1 μΜ forskolin, with different concentrations of CPA or IB-MECA.After 30 min of stimulation, the cAMP levels were determined with a LANCE cAMP kit (PerkinElmers, MA, US).
Flow Cytometry.To assess the cell surface expression level of FLAG-Nluc-A 3 R WT and mutants, HEK293T cells transiently transfected with FLAG-Nluc-A 3 R WT or mutants were analyzed with FACS in flow cytometry.After 48 h of transfection, 300,000 cells were harvested from each sample and washed twice with FACS buffer (PBS containing 1% BSA and 0.03% sodium azide).The cells were then incubated with 50 μL of FACS buffer containing 1:120 phycoerythrin (PE) anti-DYKDDDDK(FLAG) tag antibody (BioLegend, San Diego, US) in the dark for 1 h.After incubation, the cells were washed twice again and resuspended in 50 μL of FACS buffer.Analysis was performed using a BD AccuriTM C6 flow cytometer with an excitation at 488 nm and an emission wavelength at 585 nm.The resulting median intensity of cells was normalized against cell transfection with pcDNA3.1(−)as 0% and FLAG-Nluc-hA 3 R WT as 100%.
Proliferation Assay.Proliferation assays were performed using the cell counting kit-8 (CCK-8) as described previously. 35Briefly, LK-2 or NCI-H1792 cells were plated at 2500 cells/well of a clear 96-well plates in complete RPMI media and cultured for 24 h.The cells were then treated with compounds over a concentration range for 72 h.CCK-8 reagent was added, and after 3 h, plates were read using a Mithras LB 940 multimode microplate reader.
In Vitro Pharmacokinetic Assessment.The pharmacokinetic assessment of 39 was outsourced and performed by Eurofins Panlabs (MO, USA).The assessments included the determination of aqueous solubility, protein binding ability, partition coefficient, permeability across Caco-2 cells, and the metabolic stability in human liver microsomes.The aqueous solubility was determined by comparing the peak area of standard (200 μM calibration standard dissolved in a solvent made up of 60% methanol and 40% water) with the peak area of the corresponding peak in an individual buffer (PBS at pH = 7.4, simulated gastric buffer, and simulated intestinal fluid) as the method shown in ref 54.A chromatogram of 200 μM test compound along with a UV/vis spectrum with labeled absorbance maxima was generated.For protein binding assays, equilibrium dialysis was performed according to ref 55.The partition coefficient was determined from the amount of compound in the organic phase and in the aqueous phase.The amount of compound in buffer was determined as the combined, volumecorrected, and weighted areas of the corresponding peaks in the aqueous phases of three organic-aqueous samples of different compositions following ref 55.In the Caco-2 bidirectional permeability assay, the compounds were tested according to ref 56 in the human colon carcinoma cell line Caco-2.For the intrinsic clearance, the remaining tested compounds in the human liver microsomes (0.1 mg/ mL) were quantified using HPLC-MS after 0, 15, 30, 45, and 60 min based in ref 57.
Data Analysis.All the assay data was analyzed with Prism 9.3.1 (Graphpad, San Diego, CA).The affinity (pK i ) of the potential antagonists in NanoBRET ligand binding assay was determined by fitting the baseline-corrected BRET ratio response curve with the "one-site K i model" based on the Cheng and Prusoff equation 58 with both the concentration (HotNM) and K d (HotKDNM) values of the "Hot ligand" CA200645 set to 5 nM for hA 3 R and HotNM/HotKDNM as 70 nM/100 nM or 100 nM/300 nM for CA200623 at hA 3 R or rA 3 R.For the A 3 R mutants, radioligandNM/HotKDNM was input as the corresponding K d values shown in Table 4. Receptor binding kinetics was determined as described previously 25,32 using the Motulsky and Mahan method 59 (built into Prism 9.3.1) to determine the test compound association rate constant and dissociation rate constant using rate constants previously described. 25In the cAMP competition experiments, the responses were normalized to the response of 100 μM forskolin as maximum and fitted with a three-parameter logistics equation.pK B values for the potential antagonists were determined based on eq 1 where A′ and A are the EC 50 of the response induced by NECA with the presence of the antagonist or DMSO control and K B = the affinity of the antagonist used. 58ll the statistical significance (*p < 0.05) was calculated by nonparametric Kruskal−Wallis test or one-way ANOVA with a Dunnett's multiple comparison test and determined as described in ref 60.
The data analysis of in vitro pharmacokinetic assessment was performed by Eurofins Panlabs (MO, USA).In the protein binding assay, % protein binding and % recovery were determined using eqs 2 and 3 % protein binding area area area 100 % recovery area area area 100 where area = peak area of the analyte in the protein matrix(p), buffer(b), and control sample(c).The apparent permeability coefficient (P app ) and % recovery of the test compounds were calculated using eqs 4 and ( 5) where V R/D is the volume of the receiver/donor chamber.C R/D,end is the concentration of the test compound in the receiver/donor chamber at the end time point, Δt is the incubation time, and A is the surface area of the cell monolayer.C R/D,mid is the calculated midpoint concentration of the test compound in the receiver/donor side.C D0 is the concentration of the test compound in the donor sample at time zero.For the intrinsic clearance, the half-life (t 1/2 ) was determined from the slope of the initial linear range of the logarithmic curve of compound remaining (%) against time, assuming the first-order kinetics.Also, the apparent intrinsic clearance (CLint) was calculated using eq 6 Computational Medicinal Chemistry.Preparation of Model of the Unresolved Inactive hA 3 R. Residues are described by their amino acid identity (single letter code) and position (amino acid number) within the specific GPCR with the Ballesteros and Weinstein numbering, 61 a scheme for class A GPCRs, whereby X.50 represents the defined centrally conserved residue on helix X, in superscript.Αll His residues were protonated on the Nε. 62e used for the inactive hA 3 R a ML-based model derived from GPCRdb web-tool 43 that contains predictions for GPCRs in active and inactive forms via the advanced multistate AF2 method 41,42 of hA 3 R.
We revised this model of the inactive state of hA 3 R by changing the orientation of R173 5.34 , M172, 5.33 and M174 5.35 as we previously described. 40e superimposed the experimental crystal structure ZM241385� A 2A R complex (PDB ID 3EML) 63 to our revised 40 ΑF2 model of WT hA 3 R model N(1.32)−H (7.75).(Residue numbers in parentheses refer to the Ballesteros−Weinstein numbering. 64) Then, the A 2A R protein (PDB ID 3EML) 63 and the crystal waters were removed resulting in the AF2 model of WT hA 3 R in complex with ZM241385 which was used as a template for the docking calculations.The model of hA 3 R in complex with ZM241385 was optimized using the Protein Preparation Wizard in Schrodinger suite 2021 (Protein Preparation Wizard; Epik, Schrodinger, LLC, New York, NY, 2021) 65 as we previously described. 32olecular Docking Calculations.Ligands 37-61 and 74-77 preparation was achieved as described in ref 32.Molecular docking calculations were performed using the induced-fit docking protocol of Schrodinger suite 2021 (Induced-fit Docking, Schrodinger, LLC, New York, NY, 2021) in a standard protocol (standard precision) which allows flexibility of both the ligand and the entire binding site.The AF2 model of the WT hA 3 R model in complex with ZM241385 was used as a template structure.Thus, the grid boxes for the binding site were built considering the coordinates of ZM241385.Docking of compounds 37-61 and 74-77 was performed using a softened potential in which the van der Waals scaling factor was set at 0.5 for both receptor and ligand.The Prime refinement step was set on side chain prediction of amino acid residues within 5 Å of the ligand.Subsequently, a minimization of the same set of residues and the ligand for each protein/ligand complex pose was performed.After this stage, any receptor structure in each pose reflects an induced fit to the ligand structure and conformation.For each ligand docked, a maximum of 20 poses was retained.The binding conformations of compounds 37-61 and 74-77 were analyzed, and the top-scoring docking poses were used for the MD simulations to investigate the binding profile of the tested compounds to the inactive hA 3 R.
MD Simulations.Each complex of ligands K18, 37−39, 56, 57, and 60 with hA 3 R from docking calculations was inserted in a preequilibrated hydrated POPC membrane bilayer according to the Orientation of Proteins in Membranes (OPM) database. 66The orthorhombic periodic box was set 12 Å away from the protein, and the 10 × 10 × 18 Å box consisted ca.130 lipids and 13,000 TIP3P water molecules, 67 using the System Builder utility of Desmond v4.9 (Schrodinger Release 2021-1: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2021.Maestro-Desmond Interoperability Tools, Schrodinger, New York, NY, 2021).Sodium and chloride ions were added randomly in the water phase to neutralize the systems and reach the experimental salt concentration of 0.150 M NaCl.The total number of atoms of the complex was approximately 75,000, and the simulation box dimensions was 71 × 81 × 105 Å 3 .−72 Partial charges for ligands were obtained using RESP 73 fitting of the electrostatic potentials calculated with Gaussian03 74 at the Hartree−Fock/6-31G* 75 level of theory and the antechamber of AmberTools22. 76D simulation protocol starts with energy minimization by applying 2500 steps of steepest descent to remove bad contacts and 7500 steps of conjugated gradient minimization in the presence of a harmonic restraint with a force constant of 5 kcal mol −1 Å −2 on all atoms of protein and ligand and a nonbonded cutoff of 12.0 Å.The next stage in the MD simulation protocol is to allow the system to heat up from 0 to 310 K using the Langevin thermostat (dynamics) 77 for temperature control, as implemented in the Amber22 program, 78 employing a Langevin collision frequency of 2.0 ps.Heating was accomplished in two consecutive steps in the presence of a harmonic restraint with a force constant of 10 kcal mol −1 Å −2 on all membrane, protein, and ligand atoms.In the first step, systems were heated to 100 K in a NVT of 50 ps length.In the second step, the temperature was raised to 310 K in a NPTγ (with γ = 10 dyn cm −1 ) simulation of 500 ps length.The Berendsen barostat 79 was used to adjust the density over the 500 ps simulation at constant pressure (NPTγ) (with γ = 10 dyn cm −1 ), with a target pressure of 1 bar and a 2 ps pressure relaxation time.Subsequently, the systems were equilibrated without restraints in a NPTγ simulation of 1 ns length with T = 310 K and γ = 10 dyn cm −1 .In the NPTγ simulations, semiisotropic pressure scaling to p = 1 bar was applied using a pressure relaxation time of 1.0 ps.The temperature of 310 K was used in MD simulations to ensure that the membrane state is above the main phase transition temperature of 298 K for POPC bilayers. 80onds involving hydrogen atoms were constrained by the SHAKE algorithm, 81 and a time step of 2 fs was used for the integration of the equations of motion.Long-range electrostatics were calculated using the Particle mesh Ewald summation, 79 with a 1 Å grid, and short-range nonbonding interactions were truncated at 12 Å with a continuum model long-range correction applied for energy and pressure.
The equilibration phase was followed by production MD simulation for 500 ns for 7 representative ligands (K18, 37−39, 56, 57, and 60) using the same protocol as in the final equilibration step.Snapshots were recorded every 100 ps during the production phase.Within the 500 ns MD simulation time, the total energy and rmsd of the protein backbone Cα atoms reached a plateau, and the systems were considered equilibrated and suitable for statistical analysis (see Figure 1, 4, S5, and  S6).
Two MD simulation repeats were performed for each complex using the same starting structure and applying randomized velocities.The visualization of the MD simulation trajectories was performed using VMD. 82We used ptraj and cpptraj 83 of AmberTools22, 76 MDAnalysis, 84,85 and Matplotlib programs 86 and ProLIF, 87 NumPy 88 libraries to perform analysis of the MD simulations trajectories.
Particle mesh Ewald molecular dynamics (PMEMD) is the primary engine for running MD simulations with AMBER22 software. 76All the MD simulations with AMBER22 software 76 were run on GTX 3070i GPUs in lab workstations.The pmemd.CUDA executable provides the ability to use NVIDIA GPUs to run the MD simulations.
Alchemical TI/MD Binding Free Energies Calculated with the MBAR Method.For the TI/MD calculations, the relaxed complex of compound 37 at hA 3 R from the 500 ns MD simulations in a POPC lipid bilayer with the ff19sb 39 was used as the reference structure for the calculations.Thus, binding poses of ligands aligned with 37-inactive hA 3 R complexes were used as starting structures for the alchemical calculations with our optimized 40 multistate AF2 method 41,42 of hA 3 R generated from GPCRdb 43 web-tool.These alchemical perturbations are described in Table S1.We found that the final snapshots from the 2 ns TI/MD simulations matched the final snapshots of relevant ligands from the 500 ns MD simulations (compounds K18, 37−39, 56, 57, and 60); the ligand did not change its binding pose during the much longer MD simulations.Thus, the TI/MD binding free energy simulations calculated the binding free energy change of the binding poses between the examined two ligands without any of the ligands changing its conformation inside the receptor.TI/MD calculations were also performed for the ligands in solution.
The calculation of the relative binding free energies ΔΔA b,0→1 or ΔΔA b,0,1 for two ligands 0 and 1 bound to A 3 R (for the 23 pairs of ligands shown in Table S1) can be performed using the MBAR method 89 and applying a thermodynamic cycle, 90−92 i.e., using the ΔA values obtained for the alchemical transformations of the ligands in the bound (b) and the solvent (s; water) state ΔA b,0,1 and ΔA s,0,1 (s), respectively, according to eq 7 The TI estimator computes the free energy change of the transformation 0 → 1 ΔA 0→1 or ΔA 0,1 by integrating the Boltzmann averaged dU(λ)/dλ as is described in eqs 8 and 9 MBAR 89 calculates the free energy difference between neighboring intermediate states ΔA λ→λ+1 using eq 10 where w is a function of Α(λ) and Α(λ+1).The equation is solved iteratively to give the free energy change of neighboring states ΔA λ→λ+1 which via combination yield the overall free energy change.−95 Details of the TI/MD theory 17 and the TI/MD protocol 32 have been described.Experimental relative binding free energies were estimated using the experimental binding affinities pK d in Table S1 according to eq 11 Chemistry.General Information.Melting points were determined on a Buchi apparatus and are uncorrected. 1H NMR and 13 C NMR spectra were recorded on a Bruker AVANCE III 600 or a Bruker AVANCE DRX 400 instrument in deuterated solvents and were referenced to TMS (δ scale) (Figure S8).Mass spectra were recorded with a LTQ Orbitrap Discovery instrument, possessing an Ionmax ionization source.Flash chromatography was performed on Merck silica gel 60 (0.040−0.063 mm).Analytical thin-layer chromatography (TLC) was carried out on precoated (0.25 mm) Merck silica gel F-254 plates.The purity of the target derivatives (>95%) was determined on a Thermo Finnigan HPLC System (P4000 Pump, AS3000 Autosampler, UV Spectra System UV6000LP detector, Chromquest 4.1 Software); Phenomenex HYPERSIL C18-BDS (250 mm, 4.0 mm, 5 μm); mobile phase: Method A: 0.2% formic acid in water/acetonitrile; flow rate 0.8 mL/min or Method B: 1% formic acid in water/acetonitrile/methanol (9:1); flow rate 1 mL/min; column temperature 25 °C; injection volume 5 μL (Table S9 and Figure S9).(16).Sodium hydride (60% dispersion in mineral oil, 1.1 g, 27.5 mmol) was added in two portions into a suspension of 6-chloropurine (14, 3 g, 19.39 mmol) in anhydrous N,N-dimethylformamide (40 mL) at 0 °C, and this mixture was stirred at r.t.under argon for 1 h.Then, the reaction was cooled at 0 °C, iodomethane (1.8 mL, 28.86 mmol) was added, and this mixture was stirred at r.t. for 20 h.Upon completion of the reaction, the mixture was diluted with water and extracted with dichloromethane (4 × 250 mL), and the combined organic layers were extracted with brine (2 × 400 mL), dried over sodium sulfate, and evaporated.The crude mixture was purified by column chromatography using a mixture of cyclohexane/ethyl acetate as the eluent (from 40/60 up to 10/90, v/v) to provide the pure isomers 15 and 16.
General Procedure for the Preparation of the Arylamidoximes 18−30.Method A. An aqueous solution of hydroxylamine (50 wt %, 0.75 mL, 11.4 mmol) was added into a solution of the corresponding arylnitrile (4 mmol) in ethanol (20 mL), and this mixture was refluxed for 2 h.Upon completion of the reaction, the solvent was evaporated at half volume, and the solid was filtered under vacuum, washed with a small amount of ethanol, and air-dried.The solid product was collected and recrystallized to provide the pure amidoxime.
Method B. Hydroxylamine hydrochloride (417 mg, 6 mmol) and sodium bicarbonate (504 mg, 6 mmol) were added into a solution of the corresponding arylnitrile (4 mmol) in ethanol (20 mL), and this mixture was stirred at room temperature for 90 min and then refluxed for 2 h.Upon completion of the reaction, the solvent was evaporated, water was added into the flask, and the solid was filtered under vacuum, washed with water, and air-dried.The solid product was collected and recrystallized to provide the pure amidoxime.
General Procedure for the Preparation of the Target Derivatives 37−55.The corresponding amidoxime 18−30 (1 mmol) and triethylamine (0.15 mL, 1.1 mmol) were added into a solution of the acyl chloride 34, 35, or 36 (1 mmol) in anhydrous tetrahydrofuran (5 mL), under argon, and this reaction mixture was stirred at room temperature for 2−16 h.Upon completion of the reaction, the mixture was diluted with ethyl acetate (40 mL) and extracted with water (40 mL).The aqueous layer was extracted two more times with ethyl acetate (40 mL).The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, and concentrated under reduced pressure.The resulting crude products were recrystallized to provide the pure target derivatives 37−55.

Data Availability Statement
The following link is provided to access the starting structures (docking poses) and output frames of the complexes between our revised model of the inactive hA 3 R generated using the multistate AF2 method and ligands K18, 37−39, 56, 57, and 60 from the MD simulations: https://github.com/annachor/inactive_A3R_AF2-carbonyloxycarboximidamides_MDs.

Figure 2 .
Figure 2. Binding affinity (pK i ) of 25 heterocyclic carbonyloxycarboximidamide analogues or derivatives at hA 3 R determined in NanoBRET binding assay.5 nM CA200645 was added to HEK293 cells stably expressing Nluc-hA 3 R, interacting with Nluc and produce BRET signal.The BRET ratio values were baseline-corrected with the response induced by high-concentration (1 μM) A 3 R antagonist MRS1220.Each data point represents the mean ± SEM of at least three experiments performed in duplicates.The pK i values determined were compared with the pK i of K18 previously determined in ref 25.One-way ANOVA with Dunnett's post-test was used to determine the statistical significance (*p < 0.05) compared to the pK i of K18 with black * indicating the affinity significantly higher and the gray * indicating the significantly lower one.

Figure 3 .
Figure 3. Characterization of the seven selected compounds at all human AR subtypes in cAMP accumulation assay.CHO-K1 cells stably expressing individual AR subtypes were treated with different concentrations of NECA or vehicle (V) and 1 μM forskolin in the case of G i/o -coupled hA 1 R and hA 3 R or DMSO control in the case of G s -coupled hA 2A R and hA 2B R, as well as 10 μM test compound (red) or DMSO control (blue) for 30 min.In hA 2A R and hA 2B R, cAMP response was normalized against the response induced by 100 μM forskolin, whereas in hA 1 R and hA 3 R, responses were represented as the percentage of the inhibition of cAMP response generated by 100 μM forskolin.Vertical arrow and horizontal arrow denote the significance of the change in efficacy and potency, respectively.One-way ANOVA was performed to compare the changes between DMSO only and the presence of tested compound (*p < 0.05).All values are represented as mean ± standard error of the mean (SEM), obtained in n = 3 independent experimental repeats, conducted in duplicates.

Figure 4 .
Figure 4. 500 ns MD simulations for the complex of compounds of 37 and 39 with the WT hA 3 R using the amber ff19sb. 39(Α,D) Representative frame of the ligand inside the orthosteric binding area.(B,E) Receptor−ligand interaction frequency histograms; bars are plotted only for residues with interaction frequencies ≥0.2.Color figure in frames or bar plots: ligand is shown with pink sticks and ligand's starting position with a pink wire, receptor is shown with a white cartoon and sticks, hydrogen bonding interactions are shown with yellow dashes or bars, π−π interactions are shown with green dashes or bars; hydrophobic interactions are shown with gray bars; and water bridges are shown with blue bars.(C,F) rmsd plots of Ca carbons of the protein (gray line) and of heavy atoms of the ligand (magenta line).For MD simulations, we used a revised model of the inactive form of hA 3 R we have recently published,40 generated using the multistate AF2 method41,42 of hA 3 R generated from GPCRdb web-tool;43 the complexes of the starting structure (docking pose) and final snapshot from the MD simulations are available as pdb files (see the Ancillary Information).

Figure 5 .
Figure 5. Computed ΔΔG b , TI/MD values plotted against ΔΔ Gb,exp values estimated by the experimental binding affinities pKi (TableS1) for hA3R using NanoBRET binding assay; r: correlation coefficient, s: slope.For TI/MD simulations, we used a revised model we recently published40 of the inactive form of hA 3 R generated based on the multistate AF2 method41,42 of hA 3 R.

Figure 7 .
Figure 7. Selective inhibition of hA 3 R in nonsmall cell lung carcinoma cells inhibits proliferation.(A) Inhibition of forskolin-mediated cAMP accumulation in NCI-H1792 cells in response to CPA or IB-MECA, costimulated with DMSO (blue circles) or 10 μM 37 or 39 (red squares).(B) pEC50 and E max values for the inhibition of LK-2 and NCI-H1792 cell proliferation for 37 and 39.Statistical significance determined using an unpaired Student's t-test.

Table 1 .
Chemical Structure and Binding Affinity (pK i a ) of the Seven Novel Heterocyclic Compounds, Carbonyloxycarboximidamides (Group A) or 1,2,4-Oxadiazole Derivatives (Group D), which Displayed Equal or Increased Affinity to Their Precursor K18

Table 2 .
Kinetic Parameters of Binding for the Seven High-Affinity Novel K18 Derivatives at hA 3 R a ) and K off (k 4 ) for each compound determined using NanoBRET binding assay at Nluc-hA 3 R and fitted with the "kinetics of competitive binding model".RT as determined by 1/K off .bIndicated values from ref25.

Table 3 .
Chemical Structure and Binding Affinity of the Four Analogues Based on 37 and 39

Table 5 .
Binding Affinities of 11 Compounds Determined from Different Assays at hA 3 R Showed Good Agreement to Each Other